The long term goal of this project is to characterize recombinant variants of activated factor Villa (FVIIIa) that have increased stability resulting in improved therapeutic characteristics as agents for treatment of hemophilia A. Recombinant stabilized FVIIIa with improved therapeutic properties could improve the quality of life and long term health of numerous hemophilia A sufferers in the United States. This is potentially a very significant medical benefit. Basic knowledge of the structure and function of FVIIIa allowed us to create FVIII variants stabilized by a disulfide bond and we propose to characterize these stabilized FVIII variants with respect to functional activity, inactivation, and correction of hemophilia in vivo. The primary hypothesis is that a disulfide bond-stabilized form of FVIII and other stabilized variants of FVIII that we may develop provide FVIIIa that will last longer in vivo and thereby allow correction of the FVIII deficiency of hemophilia A with less FVIII. We also hypothesize that these variants will have a normal half-life before they are activated and that they won't create a prothrombotic state at concentrations that are effective in correcting bleeding. Further, we hypothesize that the immunogenicity of these variants will not be altered. The Specific Aims are: [1] Analyze and characterize disulfide crosslinked recombinant FVIII and FVIIIa. We will use biochemical and chemical methods to characterize variants. [2] Determine the half-life of disulfide crosslinked FVIII in vivo and the effects of FVIIIa stabilization on in vivo bleeding correction. We will characterize the properties and function of disulfide bond-stabilized FVIIIa in vivo in a mouse model of hemophilia A. [3] Characterize the thrombogenicity of disulfide crosslinked FVIII. We will use an intravital model of arterial and venous thrombosis in hemophilia A mice using video microscopy visualization of thrombus formation. [4] Characterize the immunogenicity of disulfide crosslinked FVIII. It is important that modified variants of FVIII are not more immunogenic than normal FVIII. Therefore, we will quantify immune responses in mice. Relevance - One in 5000 males suffer from hemophilia A, a deficiency of coagulation factor VIII. The purpose of this project is to develop recombinant factor VIII with improved therapeutic properties as a new treatment for hemophilia A patients. This treatment could improve the quality of life and overall health of the hemophilia A sufferers.